Process for the sterilization of biological compositions and the product thereby

ABSTRACT

The present invention concerns the product produced by inactivating extracellular or intracellular pathogenic virus in a biological composition without incurring substantial disruption or inactivation of cells and without significant loss of labile proteins or other valuable biological components also contained therein, the inactivation process comprising subjecting said composition to a virucidally effective amount of irradiation in the presence of (a) a mixture of a compound that quenches type I photodynamic reactions and a compound that quenches type II photodynamic reactions or (b) a bifunctional compound that is capable of quenching both type I and type II reactions, to thereby inactivate said virus while retaining functionality of said composition. The composition is advantageously subjected to the irradiation and the mixture of compounds or bifunctional compound in the presence of an irradiation sensitizer. Moreover, the process can be advantageously combined with a different virucidal method to enhance virus inactivation.

This application is a continuation of U.S. Ser. No. 08/653,218, filedMay 24, 1996, now U.S. Pat. No. 6,214,534, and a divisional of U.S. Ser.No. 08/364,031, filed Dec. 23, 1994, now U.S. Pat. No. 5,981,163, and acontinuation-in-part of U.S. Ser. No. 08/031,787, filed Mar. 15, 1993,abandoned, which is a divisional of U.S. Ser. No. 07/706,919, filed May29, 1991, now U.S. Pat. No. 5,232,844, which is a continuation-in-partof U.S. Ser. No. 07/524,208, filed May 15, 1990, now U.S. Pat. No.5,120,649.

GOVERNMENT RIGHTS

This work is supported in part by award No. HL 41221 from the NationalHeart, Lung and Blood Institute.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns a process for rendering a biologicalcomposition substantially free of enveloped and non-enveloped virusescontained therein without substantial disruption or inactivation ofcells contained therein and without significant loss of labile proteinsor other valuable biological components also contained therein.

2. Description of Related Art

The problems associated with the application of virucidal procedures tobiological compositions and the efforts to date to overcome theseproblems, including the application of light and chemical agents isreviewed briefly in U.S. Pat. No. 5,120,649, the disclosure of which isincorporated herein by reference. See column 1, line 27, through column4, line 41, therein.

Various photodynamic sterilization techniques have been evaluated forinactivating viruses in cellular components of blood. Although many ofthese appear promising for the treatment of red cell concentrates(Matthews et al., “Photodynamic therapy of viral contaminants withpotential for blood banking applications”, in Transfusion, 28:81-83(1988); O'Brien et al., “Evaluation of merocyanine 540-sensitizedphotoirradiation as a means to inactivate enveloped viruses in bloodproducts”, in J. Lab. Clin. Med., 116:439-47 (1990); and Horowitz etal., “Inactivation of viruses in blood with aluminum phthalocyaninederivatives”, in Transfusion, 31:102-8 (1991)), photodynamic viralinactivation methods involving solely oxygen dependent reactions have sofar proved inappropriate for the treatment of platelet concentrates(Proudouz et al., “Inhibition by albumin of merocyanine 540-mediatedphotosensitization of platelets and viruses”, in Transfusion, 31:415-22(1991), Dodd et al., “Inactivation of viruses in platelet suspensionsthat retain their in vitro characteristics: comparison ofpsoralen-ultraviolet A and merocyanine 540-visible light methods”, inTransfusion, 31:483-90 (1991); and Horowitz et al., “Inactivation ofviruses in red cell and platelet concentrates with aluminumphthalocyanine (AIPc) sulfonates”, in Blood Cells, 18:141-50 (1992)).

One of the latest developments is the use of photoactive compounds. See,e.g., U.S. Pat. No. 5,120,649 and U.S. Ser. No. 07/706,919, filed May29, 1991. Psoralen, together with UVA, has been shown to kill viruses inboth cell-containing and cell-free solutions without undue damage to thevaluable components needed for transfusion. Methylene blue, togetherwith visible light, is being used to treat whole plasma. Phthalocyaninesand other heme analogs, together with visible light, are being exploredfor treatment of red blood cell concentrates and other blood components.

Treatment with psoralens and long wavelength ultraviolet light (UVA) isknown to produce various biochemical effects including oxygenindependent interactions with nucleic acids (e.g., psoralen-DNAmonoadduct formation and DNA crosslinking) and oxygen dependentreactions of a photodynamic nature (for review, see Gasparro, F. P.(Ed.) (1988) Psoralen DNA Photobiology, Vol I, Vol II, CRC Press, BocaRoton, Fla.). In contrast to the purely photodynamic proceduresappropriate for red cells (above), the use of psoralens and UVA hasdemonstrated promise as a means of photoinactivating viral contaminantsin platelet concentrates, although in most studies (Lin et al., “Use of8-methoxypsoralen and long-wavelength ultraviolet radiation fordecontamination of platelet concentrates”, in Blood, 74:517-525 (1989);and Dodd et al., supra, aminomethyl-trimethylpsoralen (AMT)), thecombination of high levels of virus inactivation and the maintenance ofplatelet function were possible only when air was exchanged withnitrogen prior to UVA irradiation, a cumbersome procedure with inherentvariability. However, it was recently demonstrated (Margolis-Nunno etal., “Virus Sterilization in Platelet Concentrates with Psoralen and UVAin the Presence of Quenchers” Transfusion, 22:541-547 (1992)), that forthe inactivation of ≧6.0 log₁₀ cell-free vesicular stomatitis virus(VSV) by AMT and UVA, the need for oxygen depletion as a means ofprotecting platelets could be obviated by inclusion of mannitol, ascavenger (quencher) of free radicals. (The addition of quenchers oftype I (free radical mediated) or of type II (singlet oxygen mediated)photodynamic reactions is frequently used in other contexts todistinguish which active oxygen species produces a particularphotodynamic effect.) Under the conditions used in that study, i.e., 25μg/ml AMT and 30 minutes of UVA with 2 mM mannitol, the inactivation ofcell-free VSV in air was in part oxygen dependent since equivalent viruskill (≧6.0 log₁₀) with oxygen depleted required 3 to 4 times more UVAirradiation time (90 minutes to 2 hours).

However, while these methods achieved a high level of kill of cell-freelipid enveloped viruses and of actively replicating, cell-associatedvirus, non-enveloped viruses and latent cell-associated viruses were notkilled to a high extent under the conditions reported therein.Therefore, there was the need to effect the kill of these latter virusforms without causing significant damage to the desired, valuablecomponents in the biological mixture. Conditions which result in thekill of ≧10⁶ infectious doses of latent or non-enveloped virus have beenshown to modify red blood cells and platelets and result in compromisedrecovery of labile proteins such as factor VIII.

One of the most successful of the numerous methods developed toinactivate viruses in biological fluids is treatment with organicsolvents and detergents; especially treatment with tri(n-butyl)phosphate(TNBP) and non-ionic detergents such as Tween 80 or Triton X-100. See,e.g., U.S. Pat. No. 4,540,573. This method results in excellent recoveryof labile proteins, e.g., coagulation factor VIII and IX, whileachieving a high level of virus kill, e.g., the killing of ≧10⁶ to ≧10⁸ID, of enveloped viruses; however, little inactivation of non-envelopedviruses. See also, U.S. Pat. No. 4,481,189, wherein viral inactivationis by treatment with nonanionic detergent, alcohols, ethers, or mixturesthereof.

Other methods of virus inactivation commonly applied to biologicalfluids usable in a transfusion setting include treatment with heat attemperatures ≧60° C. or treatment with UVC together with B-propiolactone(B-PL). Each of these methods results either in a significant loss oflabile proteins and/or incomplete virus killing. See, e.g., Horowitz,B., Biotechnology of Blood, “Inactivation of viruses found with plasmaproteins”, Goldstein, J., ed., Butterworth-Heinemann, Stoneham, 417-432,(1991). Additionally, adoption of B-PL has been slow because of itscarcinogenicity. Newer methods intended to enhance virus safety areunder development. The use of gamma irradiation has been explored in thelaboratory, but, thus far, has not been used in the treatment of acommercially available product. See, Horowitz, B., et al., “Inactivationof viruses in labile blood derivatives 1. Disruption of lipid-envelopedviruses by tri(n-butyl)phosphate/detergent combinations”, inTransfusion, 25:516-521 (1985); and Singer et al., “PreliminaryEvaluation of Phthalocyanine Photosensitization For Inactivation OfViral Pathogens in Blood Products”, [abstract] British J. Hematology,March 23-25 (1988:Abs. 31). Filters are being developed which appear toremove ≧10⁶ ID₅₀ of each of several viruses; however, small viruses,e.g., parvovirus or Hepatitis A virus, would not be expected to beremoved completely. Moreover, it is not known whether these filters canbe commercially produced with the consistency needed for virus safety.

In spite of these advances, there continues to be a need for novelmethods that achieve a high level of kill of both enveloped andnon-enveloped viruses without significant loss of labile proteins orother valuable biological components.

SUMMARY OF THE INVENTION

The overall objective of the present invention was to achieve a highlevel of inactivation of both enveloped and non-enveloped viruses inbiological compositions without incurring substantial disruption orinactivation of cells meant to be contained therein and withoutsignificant loss of labile proteins or other valuable biologicalcomponents also contained therein. This objective was satisfied with thepresent invention, which relates generally to a process for inactivatingextracellular and intracellular virus in a biological compositionwithout incurring substantial disruption or inactivation thereof, saidprocess comprising subjecting said composition to a virucidallyeffective amount of irradiation in the presence of (a) a mixture of acompound that quenches type I photodynamic reactions and a compound thatquenches type II photodynamic reactions or (b) a bifunctional compoundthat is capable of quenching both type I and type II reactions, tothereby inactivate said virus while retaining functionality of saidcomposition. The inventive process can, thus, be used to inactivateviruses in whole blood, red blood cell concentrates and plateletconcentrates, without adversely affecting red blood cell or plateletstructure or function. Similarly, the inventive process can be used toinactivate viruses in biological compositions without incurringsubstantial inactivation of desired, soluble biological substances(e.g., coagulation factor concentrates, hemoglobin solutions) containedtherein.

In accordance with another aspect of the invention, the inventiveprocess is advantageously carried out in the presence of an irradiationsensitizer compound.

In accordance with still another aspect of the invention, the inventiveprocess is advantageously combined with a different virucidal method toenhance virus inactivation.

UV treatment alone of either plasma or AHF concentrates results in arelatively high loss of coagulation factor activity under conditionswhich kill ≧10⁵ ID₅₀ of virus; however, it has been discovered that thisloss is significantly reduced (i.e., the recovery is high) whenquenchers of photodynamic reactions are added prior to UV treatment.Compare, Murray et al., “Effect of ultraviolet radiation on theinfectivity of icterogenic plasma”, in JAMA, 157:8-14 (1955); and, morerecently, Kallenbach et al., “Inactivation of viruses by ultravioletlight” in Morgenthaler J-J ed. “Virus inactivation in plasma products”,in Cum stud Hematol Blood Transfus., 56:70-82 (1989). Thus, the combinedtreatment according to the present invention results in a very highlevel of virus kill while coagulation factor activity is retained athigh levels.

Gamma-irradiation of cellular components of blood is the technique ofchoice for the prevention of transfusion-associated (TA)graft-versus-host (GVHD) as is UVB irradiation of PCs for the preventionof HLA alloimmunization. However, some compromise of RBC (potassiumleakage upon storage) and platelet (decreased bleeding time correction)integrity appear to be inherent with current irradiation protocols(Linden, J. V. and Pisciotto, P. 1992, “Transfusion associatedgraft-versus-host disease and blood irradiation,” Trans. Med. Rev.,6:116-123). The inclusion during γ-irradiation or UVB irradiation ofquenchers (e.g., flavonoids) or quencher mixtures which scavenge bothtype I and type II photoreaction products will prevent damage to RBCsand platelets under conditions where WBCs are inactivated or otherwisealtered. Recent reports of active oxygen species as the majorcontributors to potassium leakage and red cell membrane damage incurredwith γ-irradiation (Anderson and Mintz, 1992; Sadrzadeh et al., 1992)support this theory and suggest that the addition of these quencherswill prevent K+ leakage by enhancing the nucleic acid specificity ofthis WBC inactivation procedure.

In addition, the use of γ-irradiation with quencher inclusion as anaddition to viral envelope-directed virus sterilization procedures forred blood cell concentrates (RBCCs) and platelet concentrates (PCs) willassure latent virus or provirus inactivation in contaminatinglymphocytes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 comprises four graphs depicting the results of the inactivationby AMT and UVA of VSV and bacteriophage M13 in the presence of mannitol,glycerol, or a mixture of mannitol and glycerol.

FIG. 1a depicts the results for platelet function.

FIG. 1b depicts the virus kill results for inactivation of cell-freeVSV.

FIG. 1c depicts the virus kill results for cell-associated VSV.

FIG. 1d depicts the virus kill results for bacteriophage M13.

FIG. 2 comprises two graphs depicting the results of the inactivation ofVSV by AMT and UVA in the presence of α-tocopherol phosophate,tryptophan, or a mixture of α-tocopherol and tryptophan.

FIG. 2a depicts the results for platelet function.

FIG. 2b depicts the virus kill results for VSV.

FIG. 3 comprises four graphs depicting the results of the inactivationof VSV and bacteriophage M13 by AMT and UVA in the presence of mannitol,α-tocopherol phosphate, or a mixture of mannitol and α-tocopherolphosphate.

FIG. 3a depicts the results for platelet function.

FIG. 3b depicts the virus kill results for inactivation of cell-freeVSV.

FIG. 3c depicts the virus kill results for cell-associated VSV.

FIG. 3d depicts the virus kill results for bacteriophage M13.

FIG. 4 comprises two graphs depicting the results of the inactivation ofVSV by AMT and UVA in the presence of quercetin or rutin or a mixture ofα-tocopherol phosphate and mannitol.

FIG. 4a depicts the results for platelet function.

FIG. 4b depicts the virus kill results for VSV.

FIG. 5 comprises two graphs depicting the results of the inactivation ofVSV by AMT and UVA in the presence of quercetin or rutin or mannitol.

FIG. 5a depicts the results for platelet function.

FIG. 5b depicts the virus kill results for VSV.

FIG. 6 comprises one graph depicting the effect on the recovery ofcoagulation factor VIII of the inclusion of rutin during the viralinactivation treatment of plasma by AMT and UVA.

FIG. 7 comprises a single graph depicting the influence of UVC treatmentof AHF concentrate with respect to bacteriophage M13 infectivity andFVIII recovery.

FIG. 8 comprises two graphs depicting the protective and optimalconcentration of ascorbate in the presence of constant quercetin (FIG.8a) and quercetin in the presence of constant ascorbate (FIG. 8b).

FIG. 9 comprises two graphs depicting the influence of UVC treatment inthe presence of 0.5 mM ascorbate and 0.2 mM quercetin with respect tobacteriophage M13 infectivity and FVIII recovery.

FIG. 9a depicts the results on the treatment of AHF concentrate.

FIG. 9b depicts the results on the treatment of FFP.

DETAILED DESCRIPTION OF THE INVENTION

Blood is made up of solids (cells, i.e., erythrocytes, leucocytes, andplatelets) and liquid (plasma). The cells are transfused in thetreatment of anemia, clotting disorders, infections, etc. In addition,the cells contain potentially valuable substances such as hemoglobin,and they can be induced to make other potentially valuable substancessuch as interferon, growth factors, and other biological responsemodifiers. The plasma is composed mainly of water, salts, lipids andproteins. The proteins are divided into groups called fibrinogen, serumglobulins and serum albumin. Typical antibodies (immune globulins) foundin human blood plasma include those directed against infectioushepatitis, influenza H, etc.

Blood transfusions are used to treat anemia resulting from disease orhemorrhage, shock resulting from loss of plasma proteins or loss ofcirculating volume, diseases where an adequate level of plasma proteinus not maintained, for example, hemophilia, and to bestow passiveimmunization.

With certain diseases one or several of the components of blood may belacking. Thus the administration of the proper fraction will suffice,and the other components will not be “wasted” on the patient; the otherfractions can be used for another patient. The separation of blood intocomponents and their subsequent fractionation allows the cells and/orproteins to be concentrated, thus enhancing their therapeutic use.

Cell types found in human blood include red blood cells, platelets andseveral types of leukocytes. Methods for the preparation of cellconcentrates useful in transfusion can be found in Kirk Othmer'sEncyclopedia of Chemical Technology, Third Edition, IntersciencePublishers, Volume 4, pp 25-37, the entire contents of which areincorporated by reference herein.

Proteins found in human plasma include prealbumin, retinol-bindingprotein, albumin, alpha-globulins, beta-globulins, gamma-globulins(immune serum globulins), the coagulation proteins (antithrombin III,prothrombin, plasminogen, antihemophilic factor-factor VIII,fibrin-stabilizing factor-factor XIII, fibrinogen), immunoglobins(immunoglobulins G. A, M, D, and E), and the complement components.There are currently more than 100 plasma proteins that have beendescribed. A comprehensive listing can be found in “The PlasmaProteins”, ed. Putnam, F. W., Academic Press, New York (1975).

Proteins found in the blood cell fraction include hemoglobin,fibronectin, fibrinogen, platelet derived growth factor, superoxidedismutase, enzymes of carbohydrate and protein metabolism, etc. Inaddition, the synthesis of other proteins can be induced, such asinterferons and growth factors.

A comprehensive list of inducible leukocyte proteins can be found inStanley Cohen, Edgar Pick, J. J. Oppenheim, “Biology of theLymphokines”, Academic Press, New York, (1979).

Blood plasma fractionation generally involves the use of organicsolvents such as ethanol, ether and polyethylene glycol at lowtemperatures and at controlled pH values to effect precipitation of aparticular fraction containing one or more plasma proteins. Theresultant supernatant can itself then be precipitated and so on untilthe desired degree of fractionation is attained. More recently,separations are based on chromatographic processes. An excellent surveyof blood fractionation also appears in Kirk-Other's Encylopedia ofChemical Technology, Third Edition, Interscience Publishers, Volume 4,pages 25 to 62, the entire contents of which are incorporated byreference herein.

The present invention is directed to subjecting a biological compositionsuch as whole blood, red blood cell concentrates, platelet concentrates,platelet extracts, leukocyte concentrates, semen, ascites fluid, milk,lymphatic fluid, hybridoma cell lines and products derived from any ofthe above, to irradiation in the presence of a quencher or a mixture ofquenchers.

The terms “cell-containing composition”, “biological composition”, or“biological fluid”, as used herein, are not to be construed to includeany living organism. Instead, the inventive method is intended to becarried out in an in vitro environment and the cell-containingcomposition, biological composition, or biological fluid obtained by theinventive method be an in vitro produced product, but will be usable invivo.

The present invention can be employed to treat the product of acomposition containing non-blood normal or cancerous cells or theproduct of gene splicing.

The term “irradiation” is to be construed broadly to include any form ofradiation conventionally used to inactivate cells, e.g., white bloodcells, or viruses, or parasites or other pathogenic organisms, e.g.,toxoplasma gondii, trypanosoma cruzi, plasmodium malariae, or babesiamicroti, either alone or combined with some other agent or condition.Non-limiting examples of irradiation include UV (UVA, UVB, and UVC),gamma-irradiation, x-rays and visible light.

Details on the application of radiation to effect virus inactivation arewell known to those skilled in the art. Typical radiation fluences rangefor the invention are 5-100 J/cm² (preferably 50-100 J/cm²) for UVA,0.02-2 J/cm² (preferably 0.05-0.2 J/cm²) for UVC, and 1-40 kGy forγ-irradiation. Surprisingly, it has now been discovered that virusinactivation can be advantageously enhanced if the conventionalradiation treatment is carried out in the presence of a quencher or amixture of quenchers.

Suitable quenchers of quencher mixtures are any substances known toreact with both free radicals (so-called “type I quenchers”) andreactive forms of oxygen (so-called “type II quenchers”).

Representative quenchers include unsaturated fatty acids, reducedsugars, cholesterol, indole derivatives, and the like, azides, such assodium azide, tryptophan, polyhydric alcohols such as glycerol andmannitol, thiols such as glutathione, superoxide dismutase, flavonoids,such as quercetin and rutin, amino acids, DABCO, vitamins and the like.

The quencher is used in conventional quenching amounts, but,surprisingly, when used, the overall process results in perferentialdamage to the virus but not the desired biological material.

In accordance with the present invention, superior virus kill isachieved by quenching both type I and type II photodynamic reactions,i.e., by using a mixture of type I and type II quenchers or by usingcompounds, e.g., flavonoids, that are known to quench both type I andtype II reactions. The range of virus kill is in most cases broader thanthat achieved by using type I or type II quenchers alone- even ascompared to increased concentrations of the type I or type II quencher-or by using mixtures of type I quenchers or mixtures of type IIquenchers. Moreover, this broader range of virus kill is achievedwithout sacrificing intact cell functionality or structure.

Quenchers have been used previously to enhance reaction specificity innumerous systems, including X-irradiation and light activated compounds.However, the use of quenchers with UV treatment of biological fluids,especially blood protein solutions, has not been previously reported.U.S. Pat. No. 4,946,648, the disclosure of which is incorporated hereinby reference, combines UV, solvents and detergents to treat virus spikedAHF or plasma, but the results are inferior to those achieved accordingto the present invention. The best treatment allowed for 5.4 logs ofphage Kappa inactivation, accompanied by 74% of FVIII recovery. By wayof contrast, one embodiment of the inventive process resulted in theinactivation of ≧10¹⁶ ID₅₀ of VSV, a model enveloped virus, and ≧10⁸ID₅₀ of EMCV, a model non-enveloped virus, with FVIII recovery ≧80%.

The inventive process is typically carried out over a temperature rangeof 0-42° C., preferentially 20-37° C. and most preferentially 20-25° C.The inventive process is typically carried out at pH 6.5-8, mostpreferentially 7.2-7.6. Samples are typically subjected to the inventiveprocess for a period of time of less than 24 hours; preferentially lessthan 4 hours for γ- or X-irradiation. Samples can also be treatedfrozen.

In an embodiment of the present invention, the biological composition issubjected to irradiation and the quencher or quencher mixture in thepresence of an irradiation sensitizer. In this context, suitableirradiation sensitizer compounds for use in the present inventioninclude phthalocyanines, purpurins, and other molecules which resemblethe porphyrins in structure (as described above) as well as photoactivecompounds excited by ultraviolet light (e.g., psoralen,8-methoxypsoralen, 4′-aminomethyl-4,5′,8-trimethyl psoralen, bergapten,and angelicin), dyes which absorb light in the visible spectrum (e.g.,hypericin, methylene blue, eosin, fluoresceins and flavins), and dyeswhich absorb X-irradiation (e.g. brominated psoralen, brominatedhematoporphyrin, iodinated phthalocyanine). The use of such irradiationsensitizers would be readily apparent to those skilled in the art and ispreferably substantially as described in U.S. Pat. No. 5,120,649 andU.S. Ser. No. 07/706,919, filed May 29, 1991, the disclosures of whichare incorporated herein by reference.

According to another embodiment of the invention, the treatment of thebiological composition with irradiation and quencher or quencher mixtureis combined with a second virucidal method. This second virucidal methodcan be any method used conventionally to inactivate enveloped and/ornon-enveloped viruses such as, merely for example, heat treatment, dryor otherwise, pH manipulation, treatment with lipid solvents and/ordetergents, a separate irradiation treatment, e.g., withgamma-irradiation, or treatment with chemical agents, e.g.,formaldehyde.

In a preferred embodiment, the second virucidal method is asolvent/detergent treatment such as that disclosed in U.S. Pat. No.4,540,573, the disclosure of which is hereby incorporated by reference.In this embodiment, the biological fluid is contacted with adialkylphosphate or a trialkylphosphate having alkyl groups whichcontain 1 to 10 carbon atoms, especially 2 to 10 carbon atoms.Illustrative members of trialkylphosphates for use in the presentinvention include tri-(n-butyl) phosphate, tri-(t-butyl) phosphate,tri-(n-hexyl) phosphate, tri-(2-ethylhexyl) phosphate, and tri (n-decyl)phosphate, just to name a few. An especially preferred trialkylphosphateis tri-(n-butyl) phosphate. Mixtures of different trialkylphosphates canalso be employed as well as phosphates having alkyl groups of differentalkyl chains, for example, ethyl di(n-butyl) phosphate. Similarly, therespective dialkylphosphates can be employed including those ofdifferent alkyl group mixtures of dialkylphosphate. Furthermore,mixtures of di- and trialkylphosphates can be employed.

Di- or trialkylphosphates for use in the present invention are employedin an amount between about 0.01 mg/ml and about 100 mg/ml, andpreferably between about 0.1 mg/ml and about 10 mg/ml.

The di- or trialkylphosphate can be used with or without the addition ofwetting agents. It is preferred, however, to use di- ortrialkylphosphate in conjunction with a wetting agent. Such wettingagent can be added either before, simultaneously with or after the di-or trialkylphosphate contacts the biological fluid. The function of thewetting agent is to enhance the contact of the virus in the biologicalfluid with the di- or trialkylphosphate. The wetting agent alone doesnot adequately inactivate the virus.

Preferred wetting agents are non-toxic detergents. Contemplated nonionicdetergents include those which disperse at the prevailing temperature atleast 0.1% by weight of the fat in an aqueous solution containing thesame when 1 gram detergent per 100 ml of solution is introduced therein.In particular there is contemplated detergents which includepolyoxyethylene derivatives of fatty acids, partial esters of sorbitolanhydrides, for example, those products known commercially as “Tween80”, “Tween 20” and “polysorbate 80” and nonionic oil soluble waterdetergents such as that sold commercially under the trademark “TritonX100” (oxyethylated alkylphenol). Also contemplated is sodiumdeoxycholate as well as the “Zwittergents” which are syntheticzwitterionic detergents known as “sulfobetaines” such asN-dodecyl-N,N-dimethyl-2-ammonio-1-ethane sulphonate and its congenersor non-ionic detergents such as octyl-beta-D-glucopyranoside.

The amount of wetting agent, if employed, is not crucial; for example,from about 0.001% to about 10%, preferably about 0.01 to 1.5%, can beused.

Di- and trialkylphosphates may be used in conjunction with otherinactivating agents such as alcohol or ethers with or without thecopresence of wetting agents in accordance with U.S. Pat. No. 4,481,189,the disclosure of which is incorporated by reference herein.

Treatment of biological fluids with trialkylphosphate is effected at atemperature between −5° C. and 70° C., preferably between 0° C. and 60°C. The time of such treatment (contact) is for at least 1 minute,preferably at least 1 hour and generally 4 to 24 hours. The treatment isnormally effected at atmospheric pressure, although subatmospheric andsuperatmospheric pressures can also be employed.

Normally, after the treatment, the trialkylphosphate and otherinactivating agents, for example, ether, are removed, although such isnot necessary in all instances, depending upon the nature of the virusinactivating agents and the intended further processing of thebiological fluid.

Di- or trialkylphosphate and non-ionic detergents can be removed asfollows:

(1) extraction with physiologically compatible oils (U.S. Pat. No.4,789,545);

(2) diafiltration using ether insoluble, e.g. “TEFLON”, microporousmembranes which retain the plasma proteins;

(3) absorption of desired plasma components on chromatographic oraffinity chromographic supports; and

(4) precipitation, for example, by salting out of plasma proteins.

In particular, removal from AHF can be effected by precipitation of AHFwith 2.2 molal glycine and 2.0M sodium chloride. Removal fromfibronectin can be effected by binding the fibronectin on a column ofinsolubilize gelatin and washing the bound fibronectin free of reagent.

Non-limiting examples of lipid coated, human pathogenic viruses that canbe inactivated by the present invention include vesicular stomatitisvirus (VSV), Moloney sarcoma virus, Sindbis virus, humanimmunodeficiency viruses (HIV-1; HIV-2), human T-cell lymphotorophicvirus-I (HTLV-I), hepatitis B virus, non-A, non-B hepatitis virus (NANB)(hepatitis C), cytomegalovirus, Epstein Barr viruses, lactatedehydrogenase elevating virus, herpes group viruses, rhabdoviruses,leukoviruses, myxoviruses, alphaviruses, Arboviruses (group B),paramyxoviruses, arenaviruses and coronaviruses. Non-limiting examplesof non-enveloped viruses that can be inactivated by the presentinvention include parvovirus, polio virus, hepatitis A virus, entericnon-A, non-B hepatitis virus, bacteriophage M13 and satelliteadeno-associated virus (AAV).

Cell-containing compositions to be treated according to the inventionhave ≧1×10⁸ cells/ml, preferably ≧1×10⁹ cells/ml and most preferably≧1×10¹⁰ cells/ml. Furthermore, cell-containing compositions to betreated according to the invention have preferably ≧4 mg/ml protein andmore preferably ≧25 mg/ml protein and most preferably 50 to 60 mg/mlprotein (unwashed cells).

Non-cell containing compositions to be treated according to theinvention have ≧0.1 mg/ml and preferably ≧5 mg/ml protein.

In the inventive process, at least 10⁴, preferably 10⁶, infectious unitsof virus parasite or other pathogen are inactivated.

The biological compositions treated according to the invention, whileinitially containing ≧1000 infectious units of virus/L, after the virushas been inactivated and treatment according to the invention has beenconducted, have, in the case of cell-containing compositions, aretention of intact cell functionality and structure of greater than70%, preferably greater than 80% and most preferably greater than 95%.In the case of biological fluids, a retention of biological activity ofgreater than 75%, preferably greater than 85%, and most preferablygreater than 95% can be achieved.

By the inactivation procedure of the invention, most if not virtuallyall of the viruses contained therein would be inactivated. A method fordetermining infectivity levels by inoculation into chimpanzees (in vivo)is discussed by Prince, A. M., Stephen, W., Bortman, B. and van denEnde, M. C., “Evaluation of the Effect of Beta-propiolactone/UltravioletIrradiation (BPL/UV) Treatment of Source Plasma on HepatitisTransmission by Factor IX Complex in Chimpanzees”, Thrombosis andHemostasis, 44: 138-142, (1980).

According to the invention, inactivation of virus is obtained to theextent of at least “4 logs”, preferably ≧6 logs, i.e., virus in thesample is totally inactivated to the extent determined by infectivitystudies where that virus is present in the untreated sample in such aconcentration that even after dilution to 10⁴ (or 10⁶), viral activitycan be measured.

The present invention describes inactivating viruses, whilesimultaneously retaining labile blood cell functional and structuralfeatures.

Functional activities of platelets are determined by their ability toaggregate in the presence of certain biological agents and theirmorphology. Structural integrity of platelets is assessed by in vivosurvival following radiolabeling with indium-ill and identification ofthe presence of specific platelet antigens.

After treatment with the photoreactive compound, excess photoreactivecompound can be removed by centrifugation, washing dialysis,and/oradsorption onto hydrophobic matrices.

In an embodiment of the present invention, the treated cell-containingfraction from the inventive process is transfused or returned to thedonor, e.g., human donor, from which the initial cell-containingfraction was derived. In this manner, the level of circulating virus inthe donor will be reduced, thus improving the donor's ability to clearvirus and/or improving the efficacy of antiviral drugs.

Factor VIII and factor IX coagulant activities are assayed bydetermining the degree of correction in APTT time of factor VIII—andfactor IX—deficient plasma, respectively. J. G. Lenahan, Philips andPhilips, clin. Chem., Vol. 12, page 269 (1966).

The activity of proteins which are enzymes is determined by measuringtheir enzymatic activity. Factor IX's activity can be measured by thattechnique.

Binding proteins can have their activities measured by determining theirkinetics and-affinity of binding to their natural substrates.

Lymphokine activity is measured biologically in cell systems, typicallyby assaying their biological activity in cell cultures.

Protein activity generally is determined by the known and standard modesfor determining the activity of the protein or type of protein involved.

The invention will now be described with reference to the followingnon-limiting examples.

EXAMPLES Materials and Methods Blood

Whole blood was typically less than 48 hours old when used. Prior touse, it was stored at 4° C.

Platelet Concentrates (PCs)

PCs, released after routine blood bank testing, were typically 24 to 48hours old when treated. Prior to treatment, the PCs were stored at 22 to24° C. in the bags (PL 732, Fenwal Laboratories, Deerfield, Ill.) inwhich they were received and constantly agitated on a platelet rotator(Helmer Labs, St. Paul, Minn.).

Psoralen Solutions

4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) was purchased from HRIAssoc. Inc., Concord, Calif. Stock solutions of AMT (4 mg/ml) wereprepared in distilled water.

Model Virus Studies

The inactivation of the following viruses was studied: vesicularstomatitis virus (VSV), a lipid enveloped, RNA virus;encephalomyocarditis virus (EMC), a protein enveloped, RNA virus; humanimmunodeficiency virus (HIV), a human, pathogenic retrovirus; hepatitisA virus, a non-enveloped, RNA virus; adeno-associated virus, anon-enveloped, DNA virus; M13, a non-enveloped bacteriophage; andpoliovirus, a non-enveloped, RNA virus.

The pHM175 strain of HAV was propagated in monolayer cultures of Africangreen monkey kidney (BS-C-1) cells as described by Jansen et al, 1988(Jansen R. W., J. E. Newbold and S. M. Lemon, Virology, 163:299-307,1988).

Quantitation of viral infectivity was based on the autoradiographicdetection of foci developed in cell sheets maintained beneath 0.5%agarose overlays following fixation of cells with 80% acetone andsubsequent staining with I-125 labelled antibody (IgG) to HAV.

Ten fold serial dilutions of HAV in MEM culture medium supplemented with2% fetal calf serum were prepared; each dilution was used to inoculateduplicate 60 mm Corning dishes of BS-C-1 cells. After 5-7 daysincubation at 35 degrees Centigrade in a humidified environment with 5%CO₂, foci derived from individual virus particle replication werevisualized, enumerated and results were expressed in terms ofradioimmunofocus forming units (RFU) of virus.

Subconfluent 293 cells were co-infected with 10 PFU of Ad5, and 10-foldserial dilutions of AAV in DMEM supplemented with 2% fetal calf serum in24 well plates. At 48 hours post infection and incubation at 37° C. in ahumidified environment with 5% CO₂, the cells were scraped from thewells, washed, denatured and hybridized to an AAV (₃₂ P) DNA probe andautoradiography as described by Carter et al, Virology, 128:505-516,1983, was carried out. Following exposure to X-ray film and counting,cpm standard curves were drawn from the known virus stock and used todetermine the concentration of AAV in each dilution of the unknownsample.

VSV was cultured in hum an A549 cells. EMC stocks were prepared in mouseL929 or human A459 cells. Poliovirus was grown in human HeLa cells.Culturing and assay procedures were similar to those described inHorowitz, B. Wiebe, M. E., Lippin, A. and Stryker, M. H., “Inactivationof viruses in Labile Blood Derivatives”, Transfusion, 1985;25:516-522.Infectivity of VSV, EMC and poliovirus was assessed by endpoint, 10-foldserial dilutions in DMEM culture medium (Gibco Laboratories, GrandIsland, N.Y.) with 10% fetal calf serum (FCS; MA Bioproducts,Walkersville, Md.). Each dilution was used to inoculate eight replicatewells of human A549 (VSV or EMC) or HeLa (poliovirus) cells in 96-wellmicrotiter plates. Virus-induced cytopathology was scored after 72 hoursof incubation at 37° C. in 5% CO₂. The reported virus titer wascalculated using the Spearman-Karber method (Spearman, C., “The Methodof Right and Wrong Cases' (‘Constant Stimuli’) Without Gauss's Formula”,Br. J. Psychol., 1908;2:227-242) and indicates the quantity of viruswhich infects 50% of the tissue culture wells (TCID₅₀).

Cell-associated VSV was prepared by incubating a confluent monolayer ofhuman A549 cells with 5 ml of 10⁷ ID₅₀/ml VSV in serum-free DMEM for 1hour at 37° C. under 5% CO₂ in 150 cm ² tissue culture flasks. Themultiplicity of infection under these conditions was approximately 2.1TCID₅₀/cell. After decanting off the liquid, the attached cells werewashed three times to remove free virus with 50 ml PBS per wash.Afterwards, 40 ml of DMEM containing 5% FCS were added, and the cellswere incubated for an additional {fraction (4 3/4)} hours. The attachedcells were washed three times with PBS and released by treatment for 10minutes with a normal saline solution containing 0.01% trypsin (CooperBiomedical, Freehold, N.J.; two times crystallized) and 5 μg/ml EDTA.The released cells were collected by centrifugation, washed three timeswith PBS and resuspended in PBS.

For assessment of virus inactivation, the virucidal reaction was stoppedby 10-fold dilution into DMEM containing 5% fetal calf serum, and thecells when present were removed by centrifugation at 1500 rpm for 10minutes. The lack of virus inactivation at this dilution or in theabsence of irradiation was confirmed for each of the inactivationconditions studied. Samples were sterile filtered (Swinnex filters,Millipore Corp., Bedford, Mass.) and frozen at −70° C. or below untilassay.

The procedures for the assessment of the inactivation of cell-associatedVSV were similar to those of cell-free VSV, except all experiments withcell-associated VSV were carried out under totally controlled asepticconditions. At the conclusion of the experiment, the infected A549 cellswere isolated by centrifugation, washed three times with PBS bycentrifugation, resuspended in 1 ml PBS and immediately assayed for VSVinfectivity by endpoint, 10-fold serial dilutions as with cell-freevirus.

Example 1 Effect of Inclusion of Type I Quenchers During Treatment of aPlatelet Concentrate with AMT and UVA

Aliquots (3 ml) from a platelet concentrate were treated with 25 μg/mlof 4′-aminomethyl-4,5′,8-trimethylpsoralen (AMT) and 7.5 mW/cm² UVA forthe times indicated in the presence or absence of various type Iquenchers. Prior to treatment, cell-free vesicular stomatitis virus(VSV), cell-associated VSV, or the non-enveloped bacteriophage M13 wereadded separately to platelet concentrate aliquots. Following treatment,samples containing virus were assayed for viral infectivity and sampleswithout virus were stored overnight and then assayed for aggregation inresponse to 20 μg/ml collagen in Biodata aggregometer. The aggregationresponse provided in the table compares the initial rate of aggregationin the treated sample to that observed in the untreated control.

The results of Example 1, shown in Table I, indicate that with the 30minute UVA irradiation time necessary for the complete inactivation ofcell-free VSV (≧6.0 log₁₀) by AMT, the addition of certain type Iquenchers (e.g., 2 mM mannitol, 4 mM glycerol) improved plateletaggregation function from about 70% to more than 90% of control levels.There was, however, little inactivation of cell-associated VSV or of M13with 30 minute treatment, whether or not type I quenchers were present.With the UVA irradiation times of 60 minutes or more that were requiredfor more than 1 log₁₀ kill of cell-associated or non-enveloped virus,platelet function was sacrificed (the rate of aggregation was 60% orless, even in the presence of type I quenchers at concentrations of upto 50 mM).

The data in Table I also indicate that the inclusion of type Iquenchers, at concentrations of up to at least 10 mM, had no apparenteffect on the inactivation of cell-free or cell-associated VSV or of M13by AMT and UVA.

TABLE I Addition of Type I Quenchers: Effects on Platelet Aggregationand Virus Kill. Quencher Aggregation Response Virus Inactivation(log₁₀)⁽³⁾ (Concen- % Control Cell-Free Cell-Asso- tration) 30⁽¹⁾ 60 90VSV ciated VSV M13 Mannitol 95 60 32 ≧6.0 1, 2⁽⁴⁾, 1, 2⁽⁴⁾, (2 mM) 3⁽⁵⁾3⁽⁵⁾ Mannitol 95 58 n.a⁽²⁾ ≧6.0 (4 mM) Mannitol 91 56 n.a. ≧6.0 1, 2⁽⁴⁾,(10 mM) 3⁽⁵⁾ Mannitol 85 55 30 5.8 (50 mM) Glycerol 90 50 n.a. ≧6.0 (2mM) Glycerol 95 55 35 ≧6.0 1, 2⁽⁴⁾, 1, 2⁽⁴⁾, (4 mM) 3⁽⁵⁾ 3⁽⁵⁾ Glycerol80 50 n.a. ≧6.0 (10 mM) Glycerol 85 50 n.a. 5.8 (50 mM) Glutathione 8060 40 ≧6.0 (2 mM) Glutathione 80 55 25 ≧6.0 (4 mM) Glutathione 75 n.a.n.a. ≧6.0 (10 mM) Superoxide 85 50 30 ≧6.0 Dismutase (20 μg/ml)Superoxide 75 45 n.a. n.a. Dismutase (100 μg/ml) none 75 52 35 ≧6.0 1,2⁽⁴⁾, 1, 2⁽⁴⁾, (---) 3⁽⁵⁾ 3⁽⁵⁾ ⁽¹⁾minutes UVA exposure; ⁽²⁾n.a. = notavailable; ⁽³⁾Unless otherwise indicated only virus results with 30 min.UVA are provided since longer treatment times compromised plateletintegrity; ⁽⁴⁾Kill results with 60 minutes UVA; ⁽⁵⁾Kill results with 90minutes UVA.

Example 2 Effect of Inclusion of Type II Quenchers During Treatment of aPlatelet Concentrate with AMT and UVA

A platelet concentrate (3 ml) was treated with 25 μg/ml AMT and 11mW/cm² UVA for the times indicated, in the presence or absence ofvarious type II quenchers. Platelet aggregation and the inactivation ofcell-free and cell-associated VSV and M13 were assayed and reported asdescribed in Example 1. The results (Table II) indicate that with 30minutes of UVA irradiation, the presence of type II quenchers decreasedthe inactivation of cell-free VSV by AMT, and this suppression of killincreased with increased concentration of the type II quencher. Sinceplatelet function was not protected by type II quencher inclusion,effective virus kill with AMT and UVA in platelet concentrates appearedto be decreased with the addition of type II quenchers.

Table II also indicates that while the inactivation of the lipidenveloped virus VSV was inhibited, kill of the non-envelopedbacteriophage M13 was unchanged by the inclusion of type II quenchers.

TABLE II Addition of Type II Quenchers: Effects on Platelet Aggregationand Virus Kill. Virus Kill (log₁₀)⁽³⁾ Agqregation Response Cell- Cell-Quencher % control free assoc. (Con. [mM]) 30⁽¹⁾ 60 90 VSV VSV M13α-tocopherol 76 55 35 4.8 phosphate (.5) α-tocopherol 75 60 40 4.0 .5,1.5⁽⁴⁾, 1, 2⁽⁴⁾, phosphate 2⁽⁵⁾ 3⁽⁵⁾ (1.0) α-tocopherol 70 50 30 3.5phosphate (2.0) Tryptophan 70 50 30 5.5 1 1 (2) Tryptophan 60 45 20 4.9(4) Tryptophan 45 20 0 4.0 .5 1 (10) Histidine 75 55 35 5.1 (5)Histidine 65 n.a⁽²⁾ n.a.⁽²⁾ 4.8 (10) none 75 52 .35 ≧6.0 1, 2⁽⁴⁾, 1,2⁽⁴⁾, (--) 3⁽⁵⁾ 3⁽⁵⁾ ⁽¹⁾minutes UVA exposure; ⁽²⁾n.a = not available;⁽³⁾unless otherwise indicated kill results only with 30 minutes of UVAare provided; ⁽⁴⁾kill results with 60 minutes UVA; ⁽⁵⁾kill results with90 minute UVA.

Example 3 Effect of Inclusion of Mixtures of Type I Quenchers DuringTreatment of a Platelet Concentrate with AMT and VUA

Platelet concentrate aliquots (3 ml) were treated with 25 μg/ml AMT and11 mW/cm² UVA for the times indicated in the absence or individualpresence of the type I quenchers mannitol (2 mM) or glycerol (4 mM), orin the presence of the mixture of these two type I quenchers. Results(FIG. 1) are shown for platelet function (FIG. 1a) and for theinactivation of cell-free (FIG. 1b) and cell-associated (FIG. 1c) VSVand M13 (FIG. 1d), which were assayed as described in Example 1.

The effects of addition of the mixture of the type I quenchers mannitoland glycerol on platelet function (FIG. 1a) and virus kill (FIGS. 1b, 1c, 1 d) are similar to the effects of addition of individual type Iquenchers as described in Example 1. Virus kill is equivalent in theabsence or presence of mixtures of type I quenchers, and plateletfunction after the 30 minute UVA treatment which yields complete kill ofcell-free VSV, is improved by type I quencher presence. However, withthe treatment times of 60 minutes or more which are necessary for morethan 1 log₁₀ kill of cell-associated virus or of M13, platelet functionis compromised whether or not type I quenchers are included, alone or incombination.

Other type I quencher mixtures, e.g., 2 mM mannitol or 4 mM glycerolcombined with 2 mM glutathione (not shown), gave equivalent results tothose obtained with the combination of mannitol and glycerol, andprovided no more protection to platelets than either of the type Iquenchers that made up the mixture.

Example 4 Effect of Inclusion of Mixtures of Type II Quenchers DuringTreatment of a Platelet Concentrate with AMT and UVA

A platelet concentrate (3 ml aliquots) was treated with 25 μg/ml AMT and11 mW/cm² UVA for 30, 60 or 90 minutes in the presence or absence of themixture of the type II quenchers α-tocopherol phosphate (1 mM) andtryptophan (5 mM), or in the individual presence of either quencher.Results (FIG. 2) for platelet function (FIG. 2a) and virus kill ofcell-free VSV (FIG. 2b), were assayed and reported as described inExample 1.

The inclusion of type II quenchers, either individually or combined withother type II quenchers, did not provide protection to platelets (FIG.2a). In addition, the presence of type II quenchers decreased the rateof kill of cell-free VSV by AMT and UVA in air, and this suppression ofkill was additive with combined type II quenchers. In 30 minutes killwas complete (≧6.0 log₁₀) with no quenchers present, while in theindividual presence of 1 mM α-tocopherol or 5 mM tryptophan kill wasonly about 4.2 log₁₀, and with the combined presence of these quencherskill was further reduced to about 2.8 log,₁₀ (FIG. 2b). Other mixturesof type II quenchers, e.g., tryptophan or α-tocopherol in combinationwith 5 mM histidine (not shown), had similar effects; they did notprotect platelet function and their combined inclusion caused a decreasein the rate of kill of cell-free VSV which was more than that of eitherof the individual quenchers. Thus, platelet function was compromisedunder all conditions of AMT and UVA treatment in the presence of type IIquenchers (alone or in combination) in which ≧6 log₁₀ of cell-free VSVwere inactivated.

Example 5 Effect of Inclusion of Mixtures of Type I and Type IIQuenchers During Treatment of a Platelet Concentrate with AMT and UVA

Platelet concentrate aliquots (3 ml) were treated with 25 μg/ml AMT and11 mW/cm² UVA for 30, 60 or 90 minutes in the presence of a mixture ofthe type I quencher mannitol (2 mM) and the type II quencherα-tocopherol phosphate (1 mM), or in the individual presence of mannitolor α-tocopherol. Platelet function (FIG. 3a) and virus kill (FIGS. 3b, 3c, 3 d) were assayed and reported as in Example 1.

With 30 minutes of irradiation, platelet function (FIG. 3a) waspreserved with either the addition of the individual type I (mannitol)quencher or with the combination of mannitol (type I) and α-tocopherolphosphate (type II). With 60 minutes or longer of UVA, the combinedpresence of mannitol and α-tocopherol phosphate improved plateletaggregation (from about 70% to more than 90% of the control with 60minutes of UVA), whereas the individual presence of each of thesequenchers did not. Functional results using glycerol as the type Iquencher were similar to those for mannitol combined with α-tocopherol.With all type I plus type II combinations tested, virus kill resultswith combined quenchers were the same as those for the type II quencheralone, and the rate of kill of cell-free (FIG. 3b) and cell-associated(FIG. 3c) VSV were somewhat decreased while that of M13 (FIG. 3d) wasunaffected by type II quencher addition.

With the longer irradiation times made possible by the addition of thecombination of the type I and type II quenchers, mannitol (or glycerol)and α-tocopherol, the effective range of virus kill by AMT and UVA inplatelet concentrates was increased.

Example 6 Effect of Inclusion of Compounds Which Quench Both Type I andType II Photodynamic Reactions During Treatment of a PlateletConcentrate with AMT and UVA

Platelet concentrate aliquots (3 ml) were treated with 25 μg/ml AMT andUVA (11 mW/cm²) for 30, 60, or 90 minutes in the absence or presence of0.25 mM quercetin or 0.25 mM rutin, or with the mixture of 1 mMα-tocopherol phosphate and 2 mM mannitol. Platelet function and virusinactivation were assayed and reported as in Example 1 (FIG. 4).

The effects of inclusion of the flavonoids quercetin or rutin, compoundsknown to efficiently quench both type I and type II photodynamicreactions, were similar to those obtained when mixtures of type I andtype II quenchers were included during treatment. The rate of kill ofVSV (FIG. 4b), but not that of M13 (not shown, see FIG. 3d), decreased,and platelet function (FIG. 4a) was protected, even with the longerirradiation times required for complete kill of cell-free VSV.

Example 7 Flavonoid Effects on Platelet Aggregation

Collagen induced aggregation is a sensitive indicator of plateletfunction and results with collagen usually correlate well with those forplatelet morphology, activation and thromboxane and ATP release, as wellas aggregation induced by other agonists. The aggregation response(extent and initial rate of aggregation as compared to the untreatedcontrol) to collagen following treatment with 25 μg/ml AMT and 90minutes UVA are provided (Table III). Table III indicates that each ofthe flavonoids tested protected the collagen induced aggregationresponse.

TABLE III Effects of flavonoid addition on the platelet aggregationresponse following treatment with AMT and UVA. % Untreated Control-Extent/Rate Flavonoid Glycon of Aggregation Response to Collagen⁽¹⁾Quencher Form untreated treated none — 100/100 44/24 quercetin — 100/10098/98 chrysin — 100/100 90/85 rutin + 100/100 100/100 hesperidin + 98/90100/86  naringin + 90/83 78/61 ⁽¹⁾prior to overnight storage, sample wastreated with 25 μg/ml AMT and 90 minutes of UVA in the absence orpresence of flavonoid indicated.

(1) prior to overnight storage, sample was treated with 25 μg/ml AMT and90 minutes of UVA in the absence or presence of flavonoid indicated.

Example 8 Effect of Inclusion of Compounds Which Quench Both Type I andType II Photodynamic Reactions During Treatment of a PlateletConcentrate with UVA and Increasing Concentrations of AMT

Because only 3 log₁₀ of cell-associated VSV were inactivated using anAMT concentration of 25 μg/ml, the effects of higher concentrations ofAMT were assessed with and without the addition of various quenchers.Platelet concentrate aliquots (3 ml) were treated with UVA (11 mW/cm²)for 90 minutes in the presence of AMT at concentrations of 25, 50 or 100μg/ml, with out without the addition of 0.7 mM rutin, 0.7 mM quercetinor 2 mM mannitol. Platelet aggregation in response to collagen (FIG. 5a)and the inactivation of cell-associated VSV (FIG. 5b) were assayed andreported as described in Example 1.

On treatment of platelets with 90 minutes of UVA and 25 μg/ml AMT theaggregation response to collagen was only about 35% of the control and,as noted above, the addition of mannitol did not protect plateletfunction with this irradiation treatment. Aggregation was furtherdecreased (to less than 30% with 50 μg/ml and to no response at all with100 μg/ml AMT) with increasing AMT concentration whether or not mannitolwas present. When 0.7 mM rutin was included during treatment,aggregation function was increased to more than 80% of the control withall of the AMT concentrations tested (FIG. 5a). Cell-free VSV (notshown) was completely inactivated under all these treatment conditions,and with 100 μg/ml AMT and 0.7 mM rutin (or quercetin) present theinactivation of cell-associated VSV was almost 6 log₁₀ (FIG. 5b). Thus,by the inclusion of compounds which quench both type I and type IIphotodynamic reactions (e.g., flavonoids such as rutin), duringtreatment of a platelet concentrate with psoralens and UVA, plateletaggregation function was well maintained under conditions where almost 6log₁₀ cell-associated virus were inactivated in the presence of oxygen.

Example 9 Effects of Deoxygenation as Compared to Rutin Addition DuringTreatment of a Platelet Concentrate with AMT and UVA

Platelet concentrate aliquots were treated with AMT at the concentrationindicated and 90 minutes of UVA, either in air in the absence orpresence of rutin, or with the air in the tube exchanged with acombination of nitrogen (95%) and CO ₂ (5%). Platelet aggregation wasassessed as in Example 1 after overnight storage in air. Table IV showsthat with an AMT concentration of 50 μg/ml, although both deoxygenationand rutin addition were capable of improving platelet function followingAMT/UVA treatment, results with rutin were consistent from experiment toexperiment, while those with gas exchange were more variable andfrequently showed no benefit at all.

TABLE IV Platelet aggregation following 90 minute AMT and UVA treatment.Effect of rutin vs. oxygen removal on consistency of results. AMT airair deoxygenated Expt. concentration no quencher +0.5 mM Rutin noquencher 1 50 μg/ml 32/33 100/100  95/100 2 50 μg/ml 42/31 98/95 90/85 350 μg/ml 36/18 95/90 56/36 4 50 μg/ml 70/30 96/96 91/94 5 50 μg/ml 21/13100/84   3/13

Example 10 Improved Recovery of Coagulation Factors on Treatment ofPlasma with AMT and UVA with the Inclusion of Compounds Which QuenchBoth Type I and Type II Photodynamic Reactions

Human plasma (3 ml aliquots) was treated with 25, 50, 100 or 200 μg/mlof AMT and irradiation with UVA (11 mW/cm²) for 90 minutes. Recovery ofcoagulation factor VIII (Antihemophilic Factor, AHF) in samples treatedwith or without the inclusion of 1, 2 or 5 mM rutin was compared. Theresults are shown in FIG. 6. On treatment of plasma with 25 μg/ml AMTand 90 minutes of UVA, AHF recovery in the absence of rutin was only 27%of the untreated control and this low value decreased further withincreased psoralen concentration, and with 200 μg/ml AMT treatment,recovery was only 7%. Remarkably, AHF recovery was restored to 83% orgreater when rutin was included during treatment (at concentrations of 2mM or greater for AMT concentrations of up to 100 μg/ml, or at 5 m!4with an AMT concentration of 200 μg/ml, and with ≧25 μg/ml of AMT, thesetreatment conditions were sufficient to inactivate at least 4 log 10 ofthe non-enveloped bacteriophage M13.

Thus, by the addition of rutin, a compound known to quench both type Iand type II photodynamic reactions, during treatment of plasma withpsoralens and UVA, a significant increase in coagulation factor VIIIrecovery can be obtained with oxygen present, under conditions wherenon-enveloped virus can be inactivated.

The next set of examples, which are set forth below, make reference to“solvent-detergent” and/or “SD” treatment. In each case, the protocolfor this treatment was as follows: AHF concentrates were treated with0.3% tri(n-butyl) phosphate (TNBP) and 1% Tween 80 for 6 hours at 24° C.after which the added reagents were removed, where indicated, by ionexchange chromatography. Plasma was treated with 1% TNBP and 1% TritonX-100 for 4 hours at 30° C., after which the added reagents were removedby hydrophobic chromatography on a C18-containing resin.

Example 11 UV Treatment of AHF Concentrate in the Absence of AddedQuenchers

Phage M13, a non-enveloped virus, was added to an AHF concentrate. Themixture was subjected to a varying dose of UV irradiation in a quartzflow cell by varying flow rate. The source of UV light was a BLEIT155bulb (Spectronic Co., Westbury, N.Y.). Before and after irradiation,Phage M13 infectivity was measured by plaque assay on host JM101 cells;AHF activity was measured in a clot assay. The results, shown in FIG. 7,indicate that under conditions where a 5 log₁₀ inactivation of Phage M13was achieved, factor VIII recovery was less than 50%.

Example 12 UV Treatment of Plasma in the Presence of Quercetin andAscorbate

The non-enveloped Phage M13 was added to plasma. Treatment of themixture in the presence of 0.75 mM ascorbate and 0.20 mM quercetin(final concentration) with 0.073 J/cm² to 0.134/cm² UV resulted in theinactivation of 6.2 to 6.5 log₁₀ (ID₅₀) of M13, while FVIII & FIXrecovery were 82-97% and 81-94%, as shown in Table V below:

TABLE V UV Dosage Phage Kill (log₁₀) FVlII Recovery % FIX Recovery(J/cm²) −Quencher +Quencher −Quencher +Quencher −Quencher +Quencher 0 0100 100 100 100 0.073 5.1 6.4 63 86 65.5 81.0 0.083 5.6 6.5 62 82 60.083.0 0.099 6.7 6.5 64.8 83 60 94.0 0.134 5.3 5.5 73 97 59.6 85.0

Note that factor VIII and factor IX recovery in the presence ofquenchers is significantly higher, while quencher addition had nosignificant effect on virus kill.

Example 13 UV Treatment of AHF Concentrate in the Presence of Quercetinand Ascorbate

AHF prior to the solvent-detergent inactivation step in the manufactureprocess was treated with UV at 0.086 J/cm², after adding EMC or VSV.Other conditions were the same as described in example 12. It is shownin Table VI below that ≧7.0 log₁₀ EMC and VSV were inactivated.Corresponding recovery of FVIII was 80.0% when quenchers were presentduring irradiation. This compared with 33% without quenchers.

TABLE VI Virus Kill log₁₀ −Quencher +Quencher FVTTI Yield % UV DosageEMC VSV EMC VSV −Quencher +Quencher 0 0 0 0 0 100 100 0.086 ≧7.0 ≧7.0≧7.0 >7.0 33 80

Example 14 Combined Treatment of Plasma with UV and SD

Virus was added to solvent-detergent treated plasma and treated with UVat 0.083 J/cm²; Table VII shows that EMC kill was ≧7.7, VSV ≧6.5 and AAV≧3.0, while coagulation factor recovery was generally 79-105% andfibrinogen yield was 107 to 113% when quenchers were present. Thesevalues are 30-50% better than when quenchers were not added. Again,under these conditions virus kill was unaffected.

TABLE VII +Quencher [Ascorbate Fibrinogen & Coagulation Quercetin VirusKill (log₁₀) Factor Recovery (%) UV Dosage or Rutin] EMC AAV VSV FV FVUFVIII FIX FXI FRN 0 − 0 0 0 100 100 100 100 100 100 0 + 0 0 0 100 103100 93.9 112.5 106-114 0.083 − ≧7.7 ≧3.0 ≧6.5 54 46-54 58 40 70 760.083 + ≧7.7 ≧3.0 ≧6.5 53-93 70-87 83-100 79-94 86-105 107-113

Example 15 Combined Treatment of AHF Concentrate with UV and SD

Solvent-detergent-treated Factor VIII concentrate, rehydrated in 10.0 mlwater per vial was spiked with polio virus type 2, which is anon-lipid-enveloped small marker virus. Treatment with UV at 0.083J/cm², resulted in ≧5.0 log₁₀ inactivation. FVIII yield was 81-94%. Thecorresponding values in the absence of quencher were 30-40% lower. Inanother example in which AHF concentrate spiked with HAV (Hepatitis AVirus) was treated, HAV kill was ≧4 logs while FVIII recovery wassimilar to above. The results are summarized in Table VIII.

TABLE VIII Quencher and Polio FVIII HAV UV Dosage Quencher Virus KillRecovery Kill J/cm² Concentration (log₁₀) (%) log₁₀ 0 −Quenchers 0 100 00 +Quenchers 0 100 0 0.083 +0.75 mM Ascorbate ≧5.6 80.8 ≧4.4 +0.2 mMQuercetin 0.083 +0.75 mM Ascorbate ≧5.3 93.9 ≧4.4 +0.5 mM Rutin

Example 16 Treatment of Plasma with UVC in the Presence of VaryingQuenchers

Plasma was treated with UV at 0.064 to 0.09 J/cm² in the presence ofvarious quenchers. Table IX shows the recovery of various factors andfibrinogen under the conditions of treatment. Based on the above, theretention of coagulation factors and fibrinogen activity is best when UVtreatment occurs in the presence of the flavinoids with or without addedascorbate or histidine. It is significant to note that the presence ofthese quenchers did not compromise kill of any of the marker virusestested.

TABLE IX Coagulation Factor Virus Kill (log₁₀) Recovery (%) Ouencher M13EMC VSV FV FVII FVIII FIX FXI FBN FLAVONOIDS None 5.5 ≧7.7 ≧6.5 59 67 5450 49 75 Quercetin 5.0 ≧7.7 ≧6.5 75 97 70 46 50 82 Rutin — ≧7.7 ≧5.4 75104 86 63 63 96 FLAVONOID MIXTURES Quercetin + — ≧7.7 ≧6.5 72 100 93 97107 91 Ascorbate Chrysin + — ≧6.5 ≧6.5 92 101 86 100 113 96 AscorbateQuercetin + — ≧6.0 ≧6.5 — — 105 136 — — Histidine TYPE I or TYPE IIQUENCHERS ALONE Ascorbate 4.7 ≧7.7 ≧6.5 67 97 73 54 52 86 Histidine 4.0— — — — 79 69 — — Glutathione 4.4 — — — — 78 68 — — Tryptophan 4.0 — — —— 79 69 — — Mannitol 6.6 — — — — 60 58 — — Glycerol 6.6 — — — — 49 51 —— Superoxide 3.9 — — — — 84 82 — — dismutase SCNAT 1.6 — — — — 75 81 — —

SCNAT is sodium carpryl N-acetyl tryptophan.

Example 17 Recovery of FVIII in UV Treated Plasma with VaryingConcentration of Quenchers (Ascorbate and Quercetin)

Solvent-detergent treated plasma was treated with UV at 0.0865 J/cm² inthe presence of various concentrations of quercetin (0 to 1.75 mM) whilekeeping the final concentration of ascorbate in the plasma constant at0.50 mM. Conversely, the final concentration of quercetin was keptconstant at 0.20 mM while adding various concentrations of ascorbate (0to 1.50 mM). The objective was to determine the best—optimal—levels ofeach of these compounds. The results, which are shown in FIGS. 8a and 8b, indicate that each of these stabilizers is self-limiting. Quercetinpeaked at 0.20 mM. Ascorbate has a broader maximum, at 0.75 to 1.25 mM.

Example 18 Kinetics of Virus (M13) Kill in Plasma and Plasma Derivative(Solvent Detergent Treated AHF) at Various Dosages of UV (254 nM)

Fresh frozen plasma and solvent/detergent-treated AHF were independentlyseeded with the phage M13 and then treated at various UV doses, [0 to0.6 J/cm²] in the presence of quenchers (0.75 mM ascorbate and 0.2 mMquercetin). Flow rate varied-with each UV dose. The results, shown inFIGS. 9a [AHF concentrate treated] and 9 b [FFP treated], indicate thattreatment @0.04 to 0.13 J/cm² where at least 5 log₁₀ inactivation ofphage M13 was achieved, factor VIII recovery was better than 80%.

Example 19 Treatment of a Red Cell Concentrate with X-irradiation and aBrominated Sensitizer

A red blood cell concentrate was treated with X-irradiation in thepresence of a brominated hematoporphyrin derivative. The presence of 1mm rutin reduced red cell hemolysis from 8% to less than 2%. Virus kill,as measured with M13, exceeded 5 log₁₀ in each case.

Example 20 Treatment of Plasma with Gamma-Irradiation

Fresh frozen plasma was treated with 40 kGy of gamma-irradiation. Therecovery of coagulation factor IX was 77% in the absence of rutin and90% in the presence of 2 mM rutin. The kill of VSV exceeded 5 log₁₀ ineach case.

Example 21 Quencher Enhanced Photoinactivation of Viruses

The following are additional examples supporting the conclusion thatvirus killing specificity in cell components and protein solutions byphotoactive procedures can be enhanced with either a mixture of type Iand type II quenchers or a bifunctional quencher:

PROD- LOG₁₀ VIRUS KILL QUENCHER UCT INACTIVANT QUENCHER VSV M13 BENEFITPlatelet 50 μg/ml AMT + none ≧6 3 agg response ↑ Conc. 57 J/cm² UVA 0.35mM rutin ≧6 3 ≦30→85% AHF 0.1 J/cm² UVC none ≧7 5.5 AHF recovery ↑ Conc.0.5 mM rutin + ≧7 5.5 33→94% 0.75 mM asc FFP 100 μg/ml AMT + none ≧6 5AHF recovery ↑ 57 J/cm² UVA 2 mM rutin ≧6 5 11→83% FFP 0.1 J/cm² UVCnone ≧6 6 AHF recovery ↑ 0.8 mM rutin ≧6 6 56→95% FFP 1 μM M8 + none ≧5na AHF recovery ↑ 44J/cm² vis light 40 μM quer + ≧5 na 73→84% 150 μM asc(AIPcS₄, aluminum phthalocyanine tetrasulfonate; AMT;amino-methyltrimethylpsoralen; MB, methylene blue: quer, quercetin; ascascorbate)

Given what is known, virus kill is inferred as follows:

Projected Virus Kill in S/D—UV Combined Treatment

Virus Kill (log₁₀) EMC Sinbis VSV AAV HAV Polio S/D 0 ≧8.8 ≧9.2 0 0 0 UV≧7.7 ≧8.7 ≧6.5 ≧3.0 ≧4.4 ≧5.6 Combined ≧7.7 ≧17.5 ≧15.7 ≧3.0 ≧4.4 ≧5.6

Data given for (UV) treatment was obtained in the presence of 0.75 mMascorbate and 0.20 mM Quercetin and fluence of 0.086 J/cm².

Coagulation Factor Recovery was 80-90%.

Example 22 Lymphocyte Inactivation and Preservation of RBC and PlateletIntegrity

1. Gamma-Irradiation:

a. γ-irradiation of RBCCs: RBCCs are treated with γ-irradiation at dosesof 15, 25 or 50 Gy (1 Gy=1 Gray=100 Rads) using a cobalt-60 source inthe presence or absence of 0.5, 1, or 2 mM rutin. Lymphocyteinactivation is determined by measurements of ³H thymidine uptake aftermitogen (2% PHA final concentration) stimulation. Extracellular (plasma)potassium is measured after 7 days of storage at 4° C. and compared withunirradiated controls.

Irradiated lymphocytes retained only 1.5% of their ³H thymidine uptakeafter a 15 Gy exposure and none after 50 Gy and this was independent ofthe presence or concentration of quencher (rutin). Extracellularpotassium increased with increasing irradiation dosage and was decreasedto control levels by the presence of 2 mM rutin. With 50 Gy ofγ-irradiation, lymphocytes were completely inactivated and plasma K⁺ was80 mM in the absence of quenchers and 40 mM when 2 mM rutin was presentduring treatment. Thus, quenchers can be used to increase thespecificity of gamma-irradiation of RBCCs for lymphocyte inactivation.

b. RBC samples treated first with AlPcS₄ and light, and thenγ-irradiation in the presence of flavonoids: Following treatment of anRBCC (diluted with an equal volume of PBS) with 6.5 μM AlPcS₄ with 44J/cm² visible light in the presence of 4 mM glutathione (GSH), thesamples are γ-irradiated with 25 Gy in the presence of absence of 1 mMor 2 mM rutin. Plasma potassium is measured after 2 and 7 days of posttreatment storage and compared to untreated controls and RBCs treatedwith AlPcS₄ only. (In addition, to examine the effect of rutin additionon K⁺ leakage with storage after AlPcS₄ treatment, one AlPcS₄ treatedsample which was not gamma-irradiated was stored in the presence of 2 mMrutin). Lymphocyte inactivation and K⁺ leakage were measured as in a.above.

The addition of γ-irradiation to AlPcS₄ treatment increased RBC damage(plasma K⁺ at 2 days was 75 mM with, and 60 mM without γ-irradiation)unless the flavonoid rutin was present during γ-irradiation and storage(25 mM at 2 days, 40 mM at 7 days). In addition, when post-treatmentstorage is in the presence of 2 mM rutin, RBCs show less leakage ofpotassium after AlPcS₄ treatment with or without γ-irradiation.

c. PCs: PCs are treated with γ-irradiation at doses of 15, 25 or 50 Gyusing a cobolt-60 source in the presence or absence of 1 or 2 mM rutin.Lymphocyte inactivation is determined by ³H thymidine uptake aftermitogen stimulation as in 1a. above. Platelet integrity was determinedby the aggregation response (initial rate as compared to the untreatedcontrol) to 40 μM arachidonic acid and 10 μM ADP at 1 and 3 days afterstorage.

As in RBCCs, irradiated lymphocytes in PCs retained only 1.5% of their³H thymidine uptake after a 15 Gy exposure and none after 50 Gy and thiswas independent of the presence of concentration of rutin. Plateletaggregation which decreased with increasing irradiation dosage (90% with15, 80% with 25 and 70% of the control with 50 Gy after 1 day) wasreturned to near control levels (95%0 by the presence of 2 mM rutin.With 50 Gy of γ-irradiation, lymphocytes were completely inactivated andthe rate of aggregation after 3 day storage was 50% of the control inthe absence of quenchers and 80% of the control when 2 mM rutin waspresent during treatment. Thus, quenchers can be used to increase thespecificity of γ-irradiation of PCs for lymphocyte inactivation.

2. UVB irradiation of Pcs:

PCs from dogs were irradiated with 36 mJ/cm² UVB (a dose known toprevent HLA alloimmunization; Slichter et al., 1987, Blood, 69:414-418)in the presence or absence of 2 mM rutin. Treated platelets wereradiolabeled (⁵¹chromium) and infused. Survival of UV-exposed donorplatelets was reduced to 2.5 days when treatment was in the absence ofrutin, but survival was the same as in untreated autologous dogplatelets (5 days) when irradiation was in the presence of rutin.

It will be appreciated that the instant specification is set forth byway of illustration and not limitation, and that various modificationsand changes may be made without departing from the spirit and scope ofthe present invention.

What is claimed is:
 1. A process for inactivating an extracellular orintracellular virus which may be present in a biological composition,said biological composition comprising at least one of a cell-containingbiological composition and a biological fluid composition, said processcomprising subjecting said biological composition to a virucidallyeffective amount of artificial irradiation, in the presence of (a) amixture of at least one quencher compound that quenches type Iphotodynamic reactions and at least one quencher compound that quenchestype II photodynamic reactions, or (b) a quencher compound that quenchesboth type I and type II reactions, or (c) a mixture of a quenchercompound that quenches both type 1 and type II reactions and anadditional quencher compound, whereby said quencher compound(s) arepresent at a concentration effective to substantially maintain, in thecase of said cell-containing biological composition, at least 70% ofstructural integrity of cells in said cell-containing biologicalcomposition and in the case of said biological fluid composition, atleast 75% of activity of said biological fluid composition.
 2. Theprocess according to claim 1, wherein said biological composition isexposed to a virucidally effective amount of artificial irradiation inthe presence of a mixture of at least one quencher compound thatquenches type I photodynamic reactions and at least one quenchercompound that quenches type II photodynamic reactions.
 3. The processaccording to claim 2, wherein said quencher compound that quenches typeI photodynamic reactions is at least one of mannitol, glycerol,glutathione, and superoxide dismutase, and said quencher compound thatquenches type II photodynamic reactions is at least one of α-tocopherolphosphate, tryptophan, and histidine.
 4. The process according to claim1, wherein said biological composition is exposed to a virucidallyeffective amount of artificial irradiation in the presence of a quenchercompound that quenches both type I and type II reactions.
 5. The processaccording to claim 4, wherein said quencher compound that quenches bothtype I and type II reactions is a flavonoid.
 6. The process according toclaim 5, wherein said flavonoid is selected from the group consisting ofquercetin, chrysin, catechin, rutin, hesperidin and naringin.
 7. Theprocess according to claim 5, wherein said quencher compound is presentat a concentration between about 0.1 and about 5 mM.
 8. The processaccording to claim 5, wherein said quencher compound is present in aconcentration of at least about 0.2 mM.
 9. The process according toclaim 1, wherein said biological composition contains red blood cells.10. The process according to claim 1, wherein said biologicalcomposition is selected from the group consisting of whole blood and redcell concentrates.
 11. The process according to claim 1, wherein saidstructural integrity of said red blood cells is ascertained bydetermining an amount of hemoglobin released after treatment of saidbiological composition with irradiation and said quencher compound(s), arelease of less than 30% of said hemoglobin indicating that saidstructural integrity of at least 70% of said red blood cells wasretained after said treatment.
 12. The process according to claim 1,wherein said biological composition contains platelets.
 13. The processaccording to claim 12, wherein said biological composition is a plateletconcentrate.
 14. The process according to any of claims 12, whichresults in a retention of structural integrity of at least 70% of saidplatelets, said structural integrity of said platelets being ascertainedby counting a number of platelets remaining after treatment of saidbiological composition with irradiation and said quencher compound(s), aretention of greater than 70% of said platelets indicating that saidstructural integrity of at least 70% of said platelets was retainedafter said treatment.
 15. The process according to claim 1, wherein thebiological composition comprises a cell-containing biologicalcomposition, and wherein the process results in a retention ofstructural integrity of the cells of at least 80%.
 16. The processaccording to claim 1, wherein the biological composition comprises acell-containing biological composition, and wherein the process resultsin a retention of structural integrity of the cells of at least 95%. 17.The process according to claim 1, wherein the biological compositioncomprises a biological fluid composition, and wherein the processresults in a retention of at least 85% of the activity of the biologicalfluid composition.
 18. The process according to claim 1, wherein thebiological composition comprises a biological fluid composition, andwherein the process results in a retention of at least 95% of theactivity of the biological fluid composition.
 19. The process accordingto claim 1, wherein said biological composition is devoid of cells. 20.The process according to claim 1, wherein said biological compositioncontains at least one coagulation factor.
 21. The process according toclaim 20, wherein said coagulation factor is selected from the groupconsisting of factors V, VII, VIII, IX and XI and fibrinogen.
 22. Theprocess according to claim 20, wherein said coagulation factor is factorVIII.
 23. The process according to claim 1, wherein said biologicalcomposition is subjected to irradiation and said quencher compound(s) inthe presence of an irradiation sensitizer.
 24. The process according toclaim 23, wherein said irradiation sensitizer is a psoralen.
 25. Theprocess according to claim 24, wherein said psoralen is4′-aminomethyl-4,5′,8-trimethylpsoralen.
 26. The process according toclaim 23, wherein said irradiation is UVA.
 27. The process according toclaim 23, wherein said irradiation sensitizer is a brominatedhematoporphyrin.
 28. The process according to claim 1, wherein saidbiological composition contains an extracellular or intracellular virusselected from the group consisting of vesicular stomatitis virus,encephalomyocarditis virus, human immunodeficiency virus, hepatitis Avirus, hepatitis B virus, non-A, non-B hepatitis virus, adeno-associatedvirus, M13 and polio virus.
 29. The process according to claim 1,wherein said irradiation is UV, gamma-irradiation, x-ray or visiblelight.
 30. The process according to claim 1, wherein said irradiation isUVA, UVB or UVC.